This protocol is for S. cerevisiae and is a modification of the one developed and used by Arjun Raj and his colleagues for:Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S. Imaging individual mRNA molecules using multiple singly labeled probes. Nature Methods. 2008 Oct;5(10):877–9.The critical change is in the digestion of the cell wall with Zymolyase (or Lyticase if you prefer). Insufficient digestion will prevent full probe penetration and will result in a misleading outcome that transcription appears to be 'off' in some fraction of your cells, even if all are transcribing.For the rest of the RNA-FISH protocol and for more details, please see Arjun's amazing website: https://sites.google.com/site/singlemoleculernafish.